Various producers of radiation measuring instruments using microtiter plates have changed over, because of decreasing budgets and a difficult cost structure for research, to instruments that can be used in multiple ways. The goal is to make a multipurpose instrument available to the customer for as many measuring methods as possible, in particular for measuring luminescence and fluorescence, so that it is unnecessary to procure a plurality of different individual instruments. Despite their higher price, compared to an instrument that is specialized for a particular measuring method, these multipurpose instruments are in strong demand. It is suggested that the customer by purchasing such a multipurpose instrument need not purchase individual instruments, especially since the price of the multipurpose instrument is less than the total price for dedicated instruments.
At present, there are many different instruments, ranging from the “dual label” instrument for luminescence and fluorescence measurements in the lowest price class, through “multilabel readers” for measuring fluorescence, luminescence and for photometry in the middle price class, to “high end” instruments for luminescence, fluorescence, photometry, fluorescence polarization, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), time resolved fluorescence (TRF), and liquid scintillation counting (LSC), in the most various combinations.
Unfortunately, in designing such multipurpose instruments for the types of measurement desired, so many compromises have to be made that in the end, their performance for the various measuring methods is markedly below that of the applicable special instrument.
The primary problem in the different qualities of functions of a multipurpose instrument is the different demands in measurement technology:
For fluorescence measurements, it is essential that the specimen be projected onto the detector and that the light be passed parallel through the filters. Since the detected light comes only from the specimen that is acted upon by the excitation light source, the problem of crosstalk, that is, the interfering scattering in of light from adjacent specimens, practically does not exist. The efficiency of the light measurement, in typical fluorescence measurements, need not be very high, since the intensity of the excitation light sources can be high.
The detection sensitivity for specific fluorophores is typically limited because of the nonspecific fluorescence of solvents, organic substances, and instrument components. This nonspecific fluorescence generally has very short decay times (approximately 4 ns for proteins). Increasing the sensitivity is achieved by time resolved fluorescence. To that end, fluorescence systems with longer decay times, for instance of several hundred microseconds, have been developed. The specimen is excited with a short flash of light, for instance lasting 0.4 μs, while the detector is turned off or is passivated. Only after the nonspecific fluorescence has decayed is the detector switched to be active, and the signal is integrated for approximately 1 ms. For each specimen, this sequence is repeated cyclically, for instance a thousand times. Using filters enhances the sensitivity still further.
Another variant in fluorescence measurement is fluorescence polarization (FP). This makes use of the fact that in the very brief time between the excitation of the fluorophore and the transmission of the fluorescent radiation, the molecule rotates in space, and the polarization plane is rotated along with it. Since small (or unbonded) molecules rotate faster than large (or bonded) molecules, information about the bond order can be obtained by measuring the degree of polarization. This method does not require any separation of bonded and unbonded molecules and is therefore especially well suited for being performed in a simplified way. To determine the degree of polarization, the specimen is illuminated with linearly polarized light, and the nonrotated (parallel) and rotated (orthogonal) components in the emitted fluorescent light are measured. This is done by means of two further polarization filters (analyzers).
In luminescence measurements, conversely, in which the photons are generated by a chemical reaction, the number of photons is much less than in fluorescence. These systems must therefore be optimized, for “collecting” all the photons emitted as completely as possible and for detecting them completely. These systems normally comprise optical systems, predominantly optical waveguides, which pick up the photons directly at the specimen and carry them on to the detector. In standard luminescence measurements, it is not necessary to place filters in between.
A more recent luminescence measurement method, especially for examining cell properties, for instance of proteins, is the aforementioned BRET. For these measurements, it is necessary to provide filters (emissions filters) upstream of the detector. Most producers for BRET therefore use their fluorometers, in which emissions filters are part of the equipment anyway. As in the case of all luminescence measurements, however, in BRET as well the photon emissions are tripped by a chemical reaction, and therefore only a small number of photons are present. The sensitivity of fluorometers is therefore inadequate for high-quality BRET measurements.
Prior Art
From European Patent Disclosure EP 0 803 724 A2, a multilabel measuring instrument is known which fails to overcome the above-discussed problems, primarily because the displaceable mirror block in it, designed for all kinds of measurements, prevents high-efficiency light passage for detecting weak luminescence signals. The space angle, detected by the lens, of the light projected from the specimen is small, and so only a small proportion of the photons originally transmitted reach the detector. Moreover, in this arrangement crosstalk of specimens in adjacent specimen holders of the microtiter plate is high. This makes the results of measurement wrong, if a highly luminous specimen located next to a specimen of low luminosity is so bright that too high a value is measured at the low-luminosity specimen.
From these examples it can be seen that the requirements made of a multipurpose measuring system for optimal function are manifold. For reasons of optics, geometric size, the availability of lenses with special material and a certain index of refraction, compromises had to be made if a more or less common optical path was to be used, and the overall result of these compromises is low performance.